Background: There is still a lack of effective treatments for acute ischemic stroke. Our pre-clinical studies suggest that ozone therapy administered by autologous blood transfusion is a convenient and safe treatment for ischemic stroke, and is popular with patients, but its therapeutic benefits are not clear. We hypothesized that ozone therapy administered by autologous blood transfusion for ischemic stroke is safe and effective, and propose a protocol for a prospective, multi-center, open-label, large-sample, parallel, randomized, non-blinded controlled trial. Methods/Design: This will be a multi-center, open-label, large-sample, parallel, randomized controlled trial. We intend to recruit 5,000 patients with acute ischemic stroke in 30 centers (including General Hospital of Shenyang Military Region, China). Patients will be randomly allocated to a control group (n = 2,500; conventional stroke therapy) or an ozone therapy group (n = 2,500; ozone therapy administered in addition to conventional therapy). The primary outcome will be a modified Rankin Scale score 0-2 at 90 days. Secondary outcomes will be National Institute of Health Stroke Scale score at 14 days, blood lipid and glucose concentrations and coagulation function at 14 days, and the incidence of post-stroke pneumonia, recurrent stroke and other vascular events in the first 90 days after stroke.Discussion: We hope that our results will illuminate the therapeutic benefits of ozone therapy administered by autologous blood transfusion for acute ischemic stroke.Trial registration: This trial was registered at Chinese Clinical Trial Registry (registration No. ChiCTR-ICR-15007093) on 18 September 2015.

Ischemic stroke is common, the mortality rate is high and there is a substantial burden of disability among survivors. Current effective therapies for ischemic stroke include antiplatelet drugs, thrombolysis with recombinant tissue plasminogen activator and endovascular stenting (Eneeva et al., 2015; Hill et al., 2015), but despite advances in treatment many patients do not make a full recovery. There is a huge amount of interest in finding more effective treatments for acute ischemic stroke, including non-drug treatments. Examples include hypothermia therapy, transcranial laser therapy, partial aortic occlusion and sphenopalatine ganglion stimulation, but none of these has been confirmed to be effective in a randomized, controlled clinical trial (Maksimova et al., 2015; Stanca et al., 2015). There has also been a great deal of interest in the potential benefits of ozone therapy for ischemic stroke (Carlsen et al., 2003).

In our clinical practice, we have observed that ozone therapy administered by autologous blood transfusion for ischemic stroke is convenient and safe, and is acceptable to patients. Nevertheless, the therapeutic benefits of ozone therapy for ischemic stroke have not been tested in a large clinical trial, so ozone therapy cannot yet be used with confidence in routine clinical practice. We hypothesized that ozone therapy administered by autologous blood transfusion is a safe and effective treatment for acute ischemic stroke, and plan to verify our hypothesis by a prospective, multi-center, open-label, large-sample, parallel, randomized controlled trial, which may provide new insights into the potential for ozone therapy to improve outcomes for patients with acute ischemic stroke.

Methods/Design

Study design

This will be a multi-center, open-label, large-sample, parallel, non-blinded randomized and controlled trial.

Ethics approval and informed consent

The study protocol has been approved by the ethics committee of General Hospital of Shenyang Military Region, China (approval No. k(2015)22). Written, informed consent will be obtained from participants and their family members.

Study participants

We will recruit 5,000 patients with acute ischemic stroke in 30 study centers (including General Hospital of Shenyang Military Region, China) to the trial.

Use of drugs that may influence the therapeutic effect of the study drug during the trial

Severe psychiatric disorders

Withdrawal criteria

Patients will be withdrawn from the trial if any of the following conditions occurs:

Lack of cooperation with the protocol

The researchers determine that to continue the trial will be harmful to participants

Participants have severe adverse reactions

Randomization

Randomization will be undertaken by the Department of Neurology, General Hospital of Shanyang Military Region, China, at the ratio of 1:1. All participants will be assigned to the ozone therapy group or the control group in accordance with randomized stratified block randomization: 2,500 participants in the control group will receive conventional treatment, while 2,500 participants in the ozone therapy group will receive ozone therapy and conventional treatment. Participants and therapists will be aware of the group management and quality plans. A flow chart of the trial protocol is shown in [Figure 1].

Participants in the control group will be managed according to routine clinical practice with antiplatelet drugs (aspirin or clopidogrel taken orally once a day), blood pressure and blood glucose control, circulatory and symptomatic support, and neuroprotection. If necessary, mannitol 20% will be used to decrease intracranial pressure and eliminate cerebral edema. Participants in the ozone therapy group will be subjected to the same management strategies but will also be administered ozone by autologous blood transfusion. Filtered sterile ozone will be produced by an ozone therapeutic instrument (Humares GmbH, Kanalstr.17-19 D-76356, Weingarten, Germany) and a known concentration (40 μg/mL) will be infused into a bag containing autologous blood and then transfused. Ozone therapy will be administered once or twice a day, for 30 minutes per episode for 10 days.

Adverse events will be recorded on the case report form, including those mentioned by participants, detected by researchers, or identified on physical examination, laboratory tests or other methods. Adverse events will be processed and reported according to relevant regulations. Case report forms will be included in the final clinical trial report.

We will classify the severity of adverse events as mild, moderate or severe. Mild adverse events are defined as symptoms or signs that are tolerable and can be accepted (accept). Moderate adverse events are defined as discomfort that has interfered with daily life (interfere). Severe adverse events are defined as those that are not compatible with daily work or activities (cannot be accepted). When determining the impact of adverse events, the term severity will be used to describe the functional impairment caused by the event.

Safety assessment

We will use the following judgment standards to assess adverse events: (1) certainly related, the reaction is consistent with the known type of reaction, the reaction improves after termination and recurs when the test is repeated; (2) possibly related, the reaction appears consistent with the known type of reaction, but the subject's clinical status or their treatment during the trial is a possible cause of the reaction; (3) possibly unrelated, the reaction is not totally consistent with the known type of reaction, and the subject's clinical status or their treatment during the trial is a possible cause of the reaction; (4) unrelated, the reaction is not consistent with the known type of reaction, and the subject's clinical status or their treatment during the trial is the probable cause of the reaction, the reaction resolves when the subject's status improves or other treatment stops and/or recurs when other treatment methods are repeatedly used; (5) cannot be assessed, the reaction is similar to the known type of reaction, and the subject's clinical status or their treatment during the trial probably caused the reaction.

We will also record the outcomes of adverse events. Adverse events will be categorized as having occurred, having not been detected, or having resolved after treatment.

Severe adverse events are defined as those that require hospitalization, prolong the length of hospital stay, lead to disability, impair work capacity, or cause a threat to life or death. The investigators will report all severe adverse events to the Work Safety Division of the China Food and Drug Administration, the hospital ethics committee of the main investigator, inspectors and sponsors of the clinical studies within 24 hours. The case report form for severe adverse events will record event-related information as completely and with as much detail as possible.

Data collection, management and statistical analysis

Experts in statistics will make an analysis proposal in consultation with the principal investigator. Case data obtained in the trial will be collected by an observer. Accuracy of data recorded in the case data form will be checked.

The following three data sets will be used: (1) Full Analysis Set, as close as possible to the intention-to-treat population, including all participants' data; (2) Per Protocol Set, a subset of the Full Analysis Set; which includes each subject has completely complied with the protocol, has complete main index values, and meets the inclusion criteria without meeting any of the exclusion or rejection criteria; (3) Safety Set, participants who were administered the study drug at least once and in whom at least one safety assessment was made.

Statistical analysis: Data will be analyzed using SAS 9.1.3 software (SAS Institute Inc., Cary, NC, USA). First, we will apply a statistical description of the data. For measurement data, the number of effective data, the mean, standard deviation, minimum value, lower quartile, median, upper quartile and maximum values will be reported, while for numeration data, corresponding frequencies or proportions will be reported, as percentages where appropriate.

Comparisons of baseline data between the groups will be made using the Mann-Whitney U non-parametric test and the chi-square test. A dropout analysis will be undertaken to compare the total dropout rate and the drop-out rate induced by adverse events using the chi-square test. For analysis of therapeutic effects, the main evaluation index (treatment efficacy) will be a non-inferiority test. The test assumptions are as follows: H 0: πT-πC ≤ −15%; H1: πT-πC > −15%. Confidence intervals (CIs) will be established: to reject H0 at α level, or if the lower limit of 95% CI of the efficacy difference between the two groups is > −15%, it can be considered that the therapeutic action of ozone is non-inferior. McNemar's chi-square test will be used to assess the efficiency of ozone in the adjuvant treatment of acute ischemic stroke. A blood low-density lipoprotein-cholesterol concentration < 2.57 mM 90 days after treatment will be used as an index for hyperlipidemia assessment. McNemar's chi-square test will be used to evaluate the therapeutic efficacy for hyperlipidemia. To reject H0 at α level, or if the lower limit of 95% CI of the efficacy difference between the two groups is > −15%, it can be considered that the therapeutic action of ozone is non-inferior. All statistical tests will be two-sided. A P value ≤ 0.05 will be considered statistically significant (with the exception of non-inferiority tests).

Data preservation: Investigators will store all data in a secure, locked environment for future viewing. In accordance with Chinese Good Clinical Practice, all data will be kept for at least 5 years.

Trial quality assurance and control

We will set up an Overall Steering Committee, an Executive Committee, a Data and Safety Monitoring Committee, and an Endpoint Event Committee to manage the scientific rigor, authenticity and reliability of the study.

Clinical trial protocol deviations will be defined thus: participants not fulfilling the inclusion criteria are nonetheless enrolled; participants do not give written, informed consent before or after the trial (taking into account that some participants may have lost the ability to write as a result of their stroke, and that written consent may be given on their behalf by a family member or appointed responsible adult); participants do not receive treatment according to the protocol; severe adverse reactions are missed or not detected in a timely fashion. If a protocol deviation adversely influences a subject's rights, safety or health, a protocol deviation report form will be completed immediately and submitted to the ethics committee for further consideration and management.

Discussion

The findings of several studies suggest that ozone therapy has some therapeutic benefits for patients with ischemic stroke (Anderson et al., 1990; Clavo et al., 2011; Frosini et al., 2012; Wasser, 2013; Sha and Huo, 2015), but each trial has been limited by its small sample size. Autohemotherapy has been reported to have beneficial therapeutic effects when combined with traditional Chinese medicine, rehabilitation, exercise and acupuncture in a trial of 156 elderly patients with ischemic stroke (Gu, 2012). Jia et al. (2015) have reported that ozonated autohemotherapy substantially improved neurologic function in 74 patients with stroke. These studies suggest that autohemotherapy is safe and effective. It is logical, therefore, that the effectiveness of ozone therapy administered by autologous blood transfusion for ischemic stroke should be verified by large-sample multi-center clinical trials.

In this prospective, multi-center, open-label, large-sample, randomized, controlled trial, 5,000 patients with acute ischemic stroke recruited in 30 centers will receive standard therapy, and 2,500 of these will also receive ozone therapy administered by autologous blood transfusion. The large number of cases that we anticipate will complete the trial will address the shortcomings of previous studies. The recovery of neurologic function will be evaluated using the mRS and NIHSS. We will also document changes in blood concentrations of lipids and glucose and coagulation parameters, evaluate adverse reactions, illuminate the effectiveness and safety of ozone therapy administered by autologous blood transfusion, and expand the evidence base for the treatment of acute ischemic stroke.

Trial status

Ongoing and recruiting at the time of submission.

Conflicts of interest

None declared.

Author contributions

JQ and HSC conceived and designed the protocols, wrote and revised the paper, and approved the final version of the paper for publication.